We have shown previously that rapamycin (RAP) inactivates p70 S6 kinase (p70S6K) in cardiac myocytes, thereby inhibiting alphaA1-adrenergic-mediated cardiac myocyte hypertrophy. The purpose of these experiments was to determine whether rapamycin-sensitive pathways play a role in regulation of RNA accumulation and expression of hypertrophy-associated genes in cardiac myocytes stimulated via alpha1-adrenergic receptors (alpha1-AR). To determine whether alpha1-AR-stimulated activation of p70, and increases in incorporation of labeled precursors into cardiomyocyte protein and RNA are sensitive to an inhibitor of phosphatidylinositol 3-kinase (PI3-K), LY294002 was employed. Cultured neonatal cardiac myocytes were administered phenyleprine (PE), vehicle (ascorbic acid) with and without rapamycin or LY294002 and harvested after 20 min, 2 days or 3 days. Lysates were immunoprecipitated with an antibody to p70, and the activity of the immune complexes assayed in vitro with a synthetic peptide substrate. PE stimulated the activity of p70 more than 3-fold, and this increase was sensitive to rapamycin, a specific inhibitor of p70. When administered for 3 days, PE stimulated 2-fold increases in protein content and in the incorporation of labelled phenylalanine (14-C-Phe) into myocyte protein and a 50% increase in two-dimensional cell area. Rapamycin (greater than or equal to 500 pg/ml) completely inhibited the PE-stimulated increases in protein content, incorporation of 14-C-Phe, and cell size. The inhibition of protein synthesis by rapamycin was competitively reversed by FK506, a related macrolide that antagonizes the effects of rapamycin. Two days of PE-treatment stimulated a 1.6-fold increase in total RNA yield/dish, a 2-fold increase in incorporation of 14-C-uridine into myocyte RNA, and increases in mRNA (relative to 18S) of atrial natriuretic factor (2.1-fold) and skeletal alpha-actin (2.2-fold). Rapamycin abolished the PE-stimulated increases in total RNA and incorporation of 14-C-uridine, but had no effect on the induction of hypertrophy-associated genes. LY294002, a specific inhibitor of phophatidylinositol 3-kinases (PI3-K)inhibited PE-stimulated increases in p70 activity, and incorporation of labeled precursors into myocyte protein and RNA in a dose dependent manner. These results indicate that p70 is activated by the hypertrophic agent PE, and suggest that p70 activation is required for PE-stimulated hypertrophy of neonatal cardiac myocytes in culture. Moreover, they suggest a bifurcation in the alpha1- adrenergic receptor signaling pathway leading to transcriptional and translational effectors. Finally, these data suggest that a PI3-K or PI3-K-like molecule lies on the alpha1-AR-p70 signaling pathway in cardiac myocytes.